Advanced Microscopy for Liver and Gut Ultrastructural Pathology in Patients with MVID and PFIC Caused by MYO5B Mutations

Abstract

Mutations in the actin motor protein myosinVb (myo5b) cause aberrant apical cargo transport and the congenital enteropathy microvillus inclusion disease (MVID). Recently, missense mutations in myo5b were also associated with progressive familial intrahepatic cholestasis (MYO5B-PFIC). Here, we thoroughly characterized the ultrastructural and immuno-cytochemical phenotype of hepatocytes and duodenal enterocytes from a unique case of an adult MYO5B-PFIC patient who showed constant hepatopathy but only periodic enteric symptoms. Selected data from two other patients supported the findings. Advanced methods such as cryo-fixation, freeze-substitution, immuno-gold labeling, electron tomography and immuno-fluorescence microscopy complemented the standard procedures. Liver biopsies showed mislocalization of Rab11 and bile canalicular membrane proteins. Rab11-positive vesicles clustered around bile canaliculi and resembled subapical clusters of aberrant recycling endosomes in enterocytes from MVID patients. The adult patient studied in detail showed a severe, MVID-specific enterocyte phenotype, despite only a mild clinical intestinal presentation. This included mislocalization of numerous proteins essential for apical cargo transport and morphological alterations. We characterized the heterogeneous population of large catabolic organelles regarding their complex ultrastructure and differential distribution of autophagic and lysosomal marker proteins. Finally, we generated duodenal organoids/enteroids from biopsies that recapitulated all MVID hallmarks, demonstrating the potential of this disease model for personalized medicine.

Case Report: MYO5B Homozygous Variant c.2090+3A>T Causes Intron Retention Related to Chronic Cholestasis and Diarrhea

“…However, the apical bile transporter ABCC2/MRP2 was mislocated intracellularly, leading to cholestasis in hepatocytes ( Overeem et al, 2020 ; Aldrian et al, 2021 ). In addition, truncated myoVb protein molecules were aberrantly localized in enterocytes with Rab11 and syntaxin abnormally bound to them ( Hess et al, 2021 ). The microvilli were generally shorter but focally had deeper rootlets and no brush border than the wild type, and microvillus inclusions were found sporadically in the epithelium of the duodenal biopsies ( Hess et al, 2021 ).…”

“…In addition, truncated myoVb protein molecules were aberrantly localized in enterocytes with Rab11 and syntaxin abnormally bound to them ( Hess et al, 2021 ). The microvilli were generally shorter but focally had deeper rootlets and no brush border than the wild type, and microvillus inclusions were found sporadically in the epithelium of the duodenal biopsies ( Hess et al, 2021 ). Aldrian et al (2021 ) proposed that these kinds of truncated proteins would cause decreased apical brush border, subapical secretory granules, and mislocated apical proteins.…”

“…The myoVb protein has five domains or regions: myosin N-terminal SH3-like, myosin motor, isoleucine-glutamine calmodulin-binding (IQ) 1-6, coiled-coil, and dilute (also called globular tail) ( https://www.uniprot.org/uniprot/Q9ULV0 ) ( Figure 1 ). Biallelic mutations in the motor domain can cause aberrant localization of bile canalicular proteins and subapical accumulation of bile salt export pumps in hepatocytes ( Overeem et al, 2020 ; Hess et al, 2021 ). However, neither a truncated myoVb without the globular tail domain nor a complete deficiency could cause mislocalization of canalicular proteins in hepatocytes ( Overeem et al, 2020 ).…”

“…However, neither a truncated myoVb without the globular tail domain nor a complete deficiency could cause mislocalization of canalicular proteins in hepatocytes ( Overeem et al, 2020 ). On the other hand, in enterocytes, truncated myoVb or total depletion of myoVb causes subapical clusters of aberrant recycling endosomes and microvillus inclusion ( Hess et al, 2021 ). In 2021, Aldrian et al reviewed and speculated that the loss of function (LoF) variation could lead to MVID, and missense or variations causing aberrant MYO5B expression would lead to PFIC or mixed MVID + PFIC ( Aldrian et al, 2021 ).…”

Structural basis of autoinhibition of the human NHE3-CHP1 complex

“…Genetic ablation of NHE3 in mice causes mild diarrhea, proximal tubular acidosis, and hypotension ( 4 ). Dysregulation of NHE3 activity has also been implicated in several human disease conditions, including cholestasis ( 5 ), cholesterol gallstone ( 6 ), cystic fibrosis ( 7 ), ulcerative colitis ( 8 ), Crohn’s disease ( 9 ), and congenital sodium diarrhea ( 10 ).…”